Microglia, the brain's resident immune cells and phagocytes, are emerging as critical regulators of developing synaptic circuits in the healthy brain. Recent studies from our lab and others indicate that microglia engulf synapses in the developing brain; however, how microglia know which synapses to target remains a major open question. Our previous work demonstrates that microglia-mediated pruning underlies developmental synaptic refinement, an essential process required for the formation of mature circuits in which weak or inappropriate synapses are eliminated and remaining connections are maintained and strengthened. We found that microglial engulfment of presynaptic inputs is activity-dependent and driven by complement molecules C1q and C3, and microglial complement receptor CR3. These molecules are innate immune eat me signals known for promoting macrophage phagocytosis of apoptotic cells or debris, and mice lacking these signals exhibit reduced microglial engulfment of synaptic inputs and impaired refinement. This suggests that microglia-mediated pruning may be analogous to the removal of non-self material by phagocytes in the immune system. However, we do not yet know how microglia precisely determine which inputs to engulf and which to avoid, an important decision regarding the specificity needed to sculpt precise, mature connections. We propose that protective don't eat me signals are required to prevent inappropriate microglial engulfment of necessary connections during synaptic refinement, just as they prevent inappropriate engulfment of healthy self-cells by phagocytes during an immune response. Our preliminary data support this hypothesis, as don't eat me signals CD47 and SIRP? are present in the developing brain and required to prevent excess microglial engulfment of synaptic inputs. We will investigate the anatomical, functional, and behavioral abnormalities in mice lacking CD47 and SIRP? to better understand the consequences of excess microglial engulfment. We will also investigate whether and how these don't eat me signals are regulated by activity to determine if they direct microglia to engulf specific synapses in an activity-dependent manner. Finally, as don't eat me signals are known to be downregulated in the brains of patients with neurodegenerative diseases, we will examine whether these molecules are dysregulated in mouse models of Huntington's disease (HD) and could thereby underlie synapse loss caused by aberrant microglial engulfment. This study would be the first to demonstrate that synaptic protection is required to prevent inappropriate microglial engulfment of necessary connections during development. This research program will provide insight not only into the mechanisms regulating microglial engulfment of specific synapses, but also into possible mechanisms underlying synapse loss in CNS neurodegenerative diseases.